Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans. Variola causes smallpox, which has a 30–40% mortality. Protocols for vaccination rely on the vaccinia virus, which has multiple potential side effects. The United States stopped vaccinating the public against smallpox in 1972, while worldwide vaccination terminated in the late 1970s. Only 10–20% of individuals previously vaccinated are still protected due to waning immunity. Public health concerns regarding the re-emergence of variola virus have led to renewed interest in the pathogenesis of smallpox. Since ethical and public health concerns preclude in vivo work on variola virus, and the World Health Organization prohibits DNA recombination studies between variola DNA fragments and other Orthopoxvirus genomes, studies of variola require indirect approaches. In vivo model systems involving orthopoxviruses do exist but are limited primarily to vaccinia, cowpox, and ectromelia viruses. The relative benign nature of other orthopoxviruses in humans may underestimate the importance of any homologous protein in the pathogenesis of smallpox.
Protocols for resuming the administration of smallpox vaccine, i.e., vaccinia virus, also have serious implications. In the last decades, the growing number of immunocompromised patients suffering from AIDS, cancer and other immunocompromising medical conditions has increased. In addition, the widespread use of immunosuppressants for organ transplant patients, the common practice of radiation and chemotherapy for treating malignancies, as well as the growing size of the aging population have also increased. Administration of the current smallpox vaccine and subsequent shedding of this virus may result in vaccinia virus infections in the population of immunocompromised individuals. Therefore, alternative smallpox vaccines or approaches to attenuate the existing vaccine are imperative at this time.
Complement regulatory proteins (CRPs), encoded by genes located in the terminal regions of orthopoxviruses, are important for viruses to evade a host-mediated complement attack (Cooper, N. R. Complement and Viruses. In: The Human complement System in Health and Disease, Vol. 1 (eds. Volanakis, J. E. & Frank, M. M.) 393–407, Marcel Dekker, Inc. NY, 1998). Virally-encoded CRPs deflect complement destruction of infected host cells and viral particles to allow for more efficient viral spread (Shchelkunov et al. (1993) FEBS Lett. 319:80–83; Lachmann and Davies (1997) Immunological Reviews 159:69–77). CRPs differ with respect to ligand specificity (C3b and/or C4b) and the mechanism of convertase inactivation. They may accelerate the normal decay of the classical and alternative pathway convertases or function as cofactors for the serine protease factor I, to enzymatically cleave the α′ chains of C3b and C4b into smaller, inactive fragments (Liszewski and Atkinson. Regulatory Proteins of Complement. In: The Human complement System in Health and Disease, Vol. 1 (eds. Volanakis, J. E. & Frank, M. M.) 149–166, Marcel Dekker, Inc. NY, 1998). Structurally, CRPs are composed of 4–56 homologous motifs termed short consensus repeats (SCR).
Vaccinia Virus Complement Control Protein (VCP) is a CRP encoded by vaccinia virus that has been shown to enhance the virulence of vaccinia in rabbit and guinea pig experimental models and causes larger lesions when injected intradermally (Isaacs et al. (1992) Proc. Natl. Acad. Sci. USA 89:628–632). VCP functions primarily as a cofactor for factor I rather than as a decay accelerator (Kotwal et al. (1990) Science 250:827–830; McKenzie et al. (1992) J. Infect. Dis. 166:1245–1250; Sahu et al. (1998) J. Immunol. 160:5596–5604). U.S. Pat. No. 5,157,110 describes the use of VCP to bind to C4b and inhibit the complement cascade. Furthermore, U.S. Pat. No. 5,843,778 discloses the use of recombinant VCP-immunoglobulin Fc region fusion protein to modulate complement activation through binding of complement components C3b and C4b.
DNA comparison studies revealed that the genomes of all variola virus strains also encode a CRP homolog consisting of four SCRs (Massung et al. (1996) Virology 221:291–300). This Smallpox Inhibitor of Complement Enzymes (SPICE) differs from the VCP amino acid sequence by 4.6%; the 11 amino acid differences are dispersed throughout SCR2, SCR3 and SCR4. WO 99/44625 discloses a SPICE polypeptide that has been generated by molecular engineering of VCP. The intended use of this SPICE protein, fused to an immunoglobulin molecule, is to modulate complement activation. Recombinant SPICE differs from VCP protein in that it is 100-fold more potent than VCP at inactivating human C3b and is more human complement-specific than is VCP (Rosengard et al. (2002) Proc. Natl. Acad. Sci. USA 99(13):8808–13).
Few options exist for the rapid detection of smallpox infection. Ropp et al. ((1995) J. Clin. Microbiol. 33:2069–76) demonstrate the use of a combined PCR amplification-endonuclease digestion of the hemagglutinin gene to identify and differentiate smallpox from other orthopoxviruses. Moreover, U.S. patent application Ser. No. 09/781,124 describes the use of monoclonal antibodies directed to vaccinia L1 R and A33R antigens to detect, prevent, and/or treat vaccinia virus infections in vitro and in vivo. Monoclonal antibodies directed to orthopoxvirus homologs of vaccinia L1R and A33R antigens are also taught.
The present invention addresses the need for improved detection, prevention, and treatment of variola and vaccinia virus infections. Antibodies directed against SPICE and VCP proteins and SPICE and VCP-specific primers for PCR amplification of the SPICE and VCP genes are provided for the detection of variola or vaccinia virus. The present invention also addresses the need for improved vaccines for the prevention of variola and vaccinia virus infections by providing antibodies directed against SPICE/VCP for passive vaccination. Furthermore, the present invention provides SPICE fusion proteins for vaccination against smallpox virus. Treatment for variola or vaccinia virus infections is provided through administration of an antibody directed against SPICE/VCP. Finally, SPICE fusion proteins are described for binding human complement components to modulate complement activation.